I. Field of the Invention
The present invention relates to a method for measuring human C-peptide and a kit therefor.
II. Description of the Related Art
Human C-peptide is a peptide consisting of 31 amino acids, and is a constituent of proinsulin which is an insulin precursor. More particularly, human C-peptide is a polypeptide which is a decomposition product released simultaneously with insulin into the blood when insulin is formed by cleavage of proinsulin by an endopeptidase. The amino acid sequences of human proinsulin and human C-peptide are shown in SEQ ID NOs: 1 and 2, respectively, in the Sequence Listing.
Human proinsulin is a polypeptide consisting of 86 amino acids, and mainly composed of insulin B chain constituted by 1st to 30th amino acids, C-peptide constituted by 33rd to 63rd amino acids, and insulin A chain constituted by 66th to 86th amino acids. Thus, C-peptide is bound to insulin B chain through the 31st amino acid Arg and 32nd amino acid Arg, and is bound to insulin A chain through the 64th amino acid Lys and 65th amino acid Arg. As mentioned above, C-peptide is released into the blood simultaneously with insulin when proinsulin is subjected to processing for yielding insulin. Thus, C-peptide serves as an index of secretion kinetics of insulin, and the kinetics of blood C-peptide can be an important index for investigation of ability to secrete endogenous insulin in patients suffering from diabetes. In fact, measurement of C-peptide is used for diagnosis and therapy of diabetes, and is useful for diagnosis of insulinoma and insulin autoimmune syndrome.
Usually, the ratio of C-peptide to proinsulin is about 1:0.6-1.3 in healthy individuals, about 1:0.3-0.5 in pyknic type individuals, and about 1:5 in patients suffering from islet cell adenoma. It has been reported that this ratio fluctuates depending on the conditions of the disease and on the diets, and C-peptide excess state and proinsulin excess state can exist (P.N.A.S., 67, 148-155, 1970). In cases where the amount of cross-reactive substance is small, large cross-reactivity of the measurement system may not be problematic very much. However, in cases where the amount of the cross-reactive substance (i.e., proinsulin in case of C-peptide) is large, and where the abundance ratio therebetween may largely fluctuate, the measured value is not reliable as an index representing the pathological or healthy conditions. For example, in cases where the blood level of C-peptide is periodically monitored for therapy, the influence by proinsulin which is a cross-reactive substance cannot be disregarded. Thus, a system for measuring C-peptide which has a low cross-reactivity to proinsulin is strongly demanded.
For measuring C-peptide, competition immunoassays using polyclonal antibodies to C-peptide have been mainly employed. For example, Japanese Patent Publication (Kokoku) No. 57-44663 discloses a competition immunoassay using radiolabeled C-peptide, and Japanese Laid-open Patent Application (Kokai) No. 1-165962 discloses a competition immunoassay using C-peptide labeled with an enzyme. However, the detection sensitivities of C-peptide by these methods are not high, the cross reactivities with proinsulin are also high, and the reproducibilities are poor, so that an improvement thereof is needed. As a method which improved these competition immunoassays, Japanese Laid-open Patent Application (Kokai) No. 4-177166 discloses a sandwich immunoassay for measuring C-peptide using a monoclonal antibody. Although detection sensitivity was improved a little by this method, the detection sensitivity is still not satisfactory. Further, cross-reactivity was not improved by this method. Thus, a method which attains low cross-reactivity does not exist so far.
Since the linear structure of C-peptide is completely included in the linear structure of proinsulin, it has been believed that it is difficult for an immunoassay for measuring C-peptide to eliminate cross-reactivity to proinsulin.
Accordingly, an object of the present invention is to provide an immunoassay for measuring human C-peptide by which the cross-reactivity to proinsulin is low, which has a high reproducibility and high detection sensitivity, as well as to provide a kit for carrying out the immunoassay.
The present inventors thought that although the linear structure of C-peptide is completely included in the linear structure of proinsulin, C-peptide may be specifically measured if an antibody which recognizes a terminal region of C-peptide is used because the terminals of C-peptide are exposed unlike proinsulin so that the structure and/or electric conditions of the terminals of C-peptide may be different from those of the corresponding regions in proinsulin. Based on this concept, the present inventors intensively studied to discover that by sandwich immunoassay using a first anti-human C-peptide antibody which recognizes an epitope existing in the region from 1st to 10th amino acid residue from the N-terminal of the human C-peptide, and a second anti-human C-peptide antibody which is immobilized on a solid support and which recognizes an epitope existing in the region from 1st to 16th amino acid residue from the N-terminal of the human C-peptide, the first and second antibodies recognizing different epitopes so that they can simultaneously bind to human C-peptide, C-peptide may be selectively measured avoiding cross-reactivity to human proinsulin, thereby completing the present invention.
That is, the present invention provides a method for measuring human C-peptide comprising the steps of:
(i) reacting human C-peptide contained in a sample, a first anti-human C-peptide antibody or an antigen-binding fragment thereof, and a second anti-human C-peptide antibody or an antigen-binding fragment thereof immobilized on a solid support to form an immune complex among three components;
(ii) separating the formed immune complex from non-reacted antibodies and/or antigen-binding fragments thereof, and sample, and
(iii) quantifying the separated immune complex;
the first antibody recognizing an epitope existing in the region from 1st to 10th amino acid residue (residues 1-10 of SEQ ID NO:2) from the N-terminal of the human C-peptide; the second antibody recognizing an epitope existing in the region from 1st to 16th amino acid residue (residues 1-16 of SEQ ID NO:2) from the N-terminal of the human C-peptide; and the first and second antibodies recognizing different epitopes so that they can simultaneously bind to the human C-peptide.
The present invention also provides a kit for measuring human C-peptide comprising:
(a) a first vessel containing a first anti-human C-peptide antibody or an antigen-binding fragment thereof, which is labeled; and
(b) a second vessel containing a solid support on which a second anti-human C-peptide antibody or an antigen-binding fragment thereof immobilized;
the first antibody recognizing an epitope existing in the region from 1st to 10th amino acid residue (residues 1-10 of SEQ ID NO:2) from the N-terminal of the human C-peptide; the second antibody recognizing an epitope existing in the region from 1st to 16th amino acid residue (residues 1-16 of SEQ ID NO:2) from the N-terminal of the human C-peptide; and the first and second antibodies recognizing different epitopes so that they can simultaneously bind to the human C-peptide.